Esterified selenium polysaccharide and preparation method and application thereof
Technical Field
The invention relates to the technical field of selenium polysaccharide, in particular to esterified selenium polysaccharide, and a preparation method and application thereof.
Background
Selenium (Se) is an essential trace element for human bodies and plays an important role in the aspects of improving the immune function of life, resisting cancer, resisting oxidation, preventing nutritional liver necrosis and the like. Selenium is also an active component of glutathione peroxidase in biological metabolism, and thus, the role of selenium on the human body is very important. However, selenium is unevenly distributed on the earth, and is deficient in about three fifths of China, while the content of selenium in common foods is extremely low, and the effect of supplementing selenium cannot be achieved generally. In nature, the forms of selenium mainly include inorganic selenium and organic selenium. Compared with inorganic selenium, the organic selenium is a selenium-containing substance which is safer and has higher activity, and is more remarkable in immune reaction stimulation than the inorganic selenium. The main source of organic selenium is natural selenium-rich biological and artificial synthesis, and among a plurality of substances rich in organic selenium, selenium polysaccharide is greatly concerned. Selenium polysaccharide, as an organic selenium compound, has the activities of both selenium and polysaccharide, and the selenized polysaccharide is more easily absorbed and utilized by organisms. In recent years, a number of selenium-enriched foods and products have appeared, of which selenium polysaccharide is the main ingredient. The preparation method of the selenium polysaccharide comprises natural selenium-containing plant polysaccharide, microorganism enrichment culture metabolism selenium polysaccharide and artificial synthesis selenium polysaccharide. Crops planted by the selenium-rich soil are rich in selenium, the product safety is good, but the selenium content is very low, and the normal metabolic balance of the necessary selenium content in blood can not be basically realized. The artificial synthesis of selenium polysaccharide is a convenient and controllable method.
At present, the selenium polysaccharide is prepared by the following three methods in domestic and foreign documents and patents: under mild conditions, selenium modification is carried out on the polysaccharide by using monomer selenium, selenious acid or sodium selenite; an intermediate with active chemical property and an acyl chloride structure is used as a selenizing reagent for modification; the functional gene containing selenium is grafted to polysaccharide molecule. The Liangshuxuan and the like adopt sodium selenate to react with lycium barbarum polysaccharide under the catalysis of glacial acetic acid; plum Shizhou and the like utilize a chemical synthesis method, grifola polysaccharide and sodium selenite are used as raw materials to prepare grifola selenium polysaccharide, and a continuous or ultrasonic-assisted chemical synthesis process is adopted, so that the defects that the selenization site is undefined, the selenization degree is uncertain, and a large amount of toxic inorganic selenium components mixed in high-molecular-weight polysaccharide cannot be completely removed are overcome. Patent CN 1560088A discloses a method for preparing selenized glucomannan, which comprises oxidizing selenium simple substance into Se under the action of oxidant6+In Se6+Adding ethanol and hydrochloric acid into the ionic water solution to obtain selenizing reaction solution, reacting the selenizing reaction solution with glucomannan to obtain selenizing glucomannan, wherein the obtained selenizing glucomannan has low selenium content, and the selenium in the derivative is Se6+(ii) a Patent ZL 88103347 discloses a method for preparing selenocarrageenan, which uses selenium powder as raw materialNitric acid is dissolved to prepare selenium liquid, Kappa-carrageenan solution is added to carry out selenylation reaction, but the selenium content of the product obtained by the method is lower (2500-. Patent ZL 200910162003.4 discloses a method for preparing selenylated artemisia desertorum polysaccharide or potentilla anserina polysaccharide by organic method, which comprises reacting the potentilla anserina polysaccharide or the artemisia desertorum polysaccharide with selenious acid chloride to obtain selenylated artemisia desertorum polysaccharide or potentilla anserina polysaccharide. However, the selenious acid chloride used in the method is difficult to synthesize and has toxicity, and is exposed in the air when being added in the reaction process, the selenious acid chloride is oxidized, and the obtained selenious acid chloride may contain impurities such as selenoyl chloride and the like.
The polysaccharide selenylation modification technology mentioned in the above patents and documents generally has the defects of complex preparation of selenium-containing intermediates, high toxicity, long reaction time, low selenium content and yield of selenylation polysaccharide, more side reactions, unclear biological effect and the like.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the esterified selenium polysaccharide and the preparation method and the application thereof, and the obtained esterified selenium polysaccharide has definite selenium binding sites, high selenium content and no toxic or side effect.
The technical scheme of the invention is as follows:
the invention provides a preparation method of esterified selenium polysaccharide, which comprises the following steps:
(1) under the protection of nitrogen at 0 ℃, 1-5 equivalent of O ═ SeCl2Dropwise adding the solution into a D-galactose allyl glycoside solution containing 1-5 equivalents of triethylamine, reacting at room temperature for 1-4 hours, pouring into a water phase, and extracting with ethyl acetate to obtain 3, 4-oxygen-ring selenic acid galactose allyl glycoside;
(2) suspending the polysaccharide in a solvent, adding NaHCO3Mixing, dropwise adding acryloyl chloride into the polysaccharide solution, maintaining the temperature of a reaction solution not to exceed 40 ℃ during dropwise adding, and after the reaction is finished, dissolving the reaction solution in water for alcohol precipitation to obtain acrylated polysaccharide;
(3) dissolving the acrylated polysaccharide in a solvent, carrying out a displacement reaction with 3, 4-O-cyclo-selenic acid galactose allyl glycoside under the action of a Ru catalyst to obtain a reaction solution, dispersing the reaction solution in a water phase, and carrying out alcohol precipitation to obtain the esterified selenium polysaccharide.
Preferably, in the step (2), the molar ratio of the polysaccharide to the acryloyl chloride is 1:1 to 50.
Preferably, in the step (2), the molar ratio of the polysaccharide to the sodium bicarbonate is 1: 5 to 50.
Further preferably, in the step (2), the reaction is completed 2 to 3 hours after the dropwise addition is stopped.
Preferably, in the step (3), the molar ratio of the acrylated polysaccharide to the 3, 4-O-cyclo-selenic acid galactose allyl glycoside is 1: 1-4.
Preferably, in the step (3), the replacement reaction is completed within 4 to 5 hours.
Preferably, in the step (1), the method further comprises a purification step after the ethyl acetate extraction: washing the extract with saturated saline solution, drying the organic phase to dryness, and separating with chromatographic column to obtain 3, 4-O-cyclo-selenic acid galactose allyl glycoside.
Preferably, the polysaccharide is a water-soluble natural polysaccharide containing a primary hydroxyl group at the 6-position.
The invention also comprises the esterified selenium polysaccharide prepared by any one of the methods, and the structural formula of the esterified selenium polysaccharide is as follows:wherein m is a selenylated saccharide unit, and n is the number of saccharide units of the natural polysaccharide.
Preferably, the esterified selenium polysaccharide has an organic selenium content of from 1 to 10 ppm.
The invention also includes the use of the esterified selenium polysaccharide for non-therapeutic purposes in improving immunity.
Compared with the prior art, the invention has the following advantages:
the method prepares the esterified selenium product with controllable selenium content by the replacement reaction of the 3, 4-oxygen-cyclo-selenic acid galactose allyl glycoside and the acrylated polysaccharide. The selenium-containing intermediate 3, 4-O-cyclo-selenious acid galactose allyl glycoside is simple to prepare, and the yield is more than 90%; the selenium-containing intermediate can be used directly without purification or purification in the preparation process, and the obtained selenium-containing intermediate has no toxicity. In the synthesis process of the esterified selenium polysaccharide, the replacement reaction conditions are simple, the reaction time is short, and the yield is high. The selenium content of the obtained esterified selenium polysaccharide can reach 1 ten thousand to 10 ten thousand ppm, and the selenium content can be adjusted.
In the esterified selenium polysaccharide molecule prepared by the invention, selenium element exists in the form of organic ester, and selenium atom is simultaneously combined with two sugar hydroxyl groups to form selenite unit with a cyclic structure. The organic compound carrier forming the esterified selenium structure is a polysaccharide substance, has the potential of improving the blood selenium content and improving various immune functions, and can obviously improve the immune capacity of a tested organism by improving the blood selenium content.
Drawings
FIG. 1 shows the maximum expression levels of cytokines IL-4 and IFN-. gamma.in example 4.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a preparation method of esterified selenium polysaccharide, which comprises the following steps:
(1) under the protection of nitrogen at 0 ℃, 1-5 equivalent of O ═ SeCl2Dropwise adding the solution into a D-galactose allyl glycoside solution containing 1-5 equivalents of triethylamine, reacting at room temperature for 1-4 hours, pouring into a water phase, and extracting with ethyl acetate to obtain 3, 4-oxygen-ring selenic acid galactose allyl glycoside;
(2) suspending the polysaccharide in a solvent, adding NaHCO3Mixing, dropwise adding acryloyl chloride into the polysaccharide solution, maintaining the temperature of a reaction solution not to exceed 40 ℃ during dropwise adding, and after the reaction is finished, dissolving the reaction solution in water for alcohol precipitation to obtain acrylated polysaccharide;
(3) dissolving the acrylated polysaccharide in a solvent, carrying out a displacement reaction with 3, 4-O-cyclo-selenic acid galactose allyl glycoside under the action of a Ru catalyst to obtain a reaction solution, dispersing the reaction solution in a water phase, and carrying out alcohol precipitation to obtain the esterified selenium polysaccharide.
Wherein, the step (1) and the step (2) are not limited in order.
The reaction process of the esterified selenium polysaccharide of the invention is as follows:
in the step of synthesizing the 3, 4-O-cyclo selenic acid galactose allyl glycoside, O is changed into SeCl under the protection of nitrogen at 0 DEG C2Dropwise adding the mixture into a D-galactose allyl glycoside solution containing triethylamine, and carrying out substitution reaction at room temperature. Among them, D-galactoallylglycoside is preferably dissolved in DMF or DMSO to form a D-galactoallylglycoside solution. In the present invention, O ═ SeCl2The amount of the triethylamine is preferably 2 to 4 equivalents, and the amount of the triethylamine is preferably 2 to 4 equivalents. In the present invention, the reaction time of the reaction is preferably 2 to 3 hours. After the reaction, the reaction solution was poured into the aqueous phase and extracted with ethyl acetate to obtain 3, 4-O-galactoallylselenite. Preferably, the method further comprises a purification step after the ethyl acetate extraction: washing the extract with saturated saline solution, drying the organic phase to dryness, and separating with chromatographic column to obtain 3, 4-O-cyclo-selenic acid galactose allyl glycoside. The purification steps can be carried out by methods customary in the art. In the invention, the organic phase is preferably dried by anhydrous sodium sulfate, the dried organic phase is preferably evaporated to dryness under reduced pressure on a rotary evaporator, and the chromatographic column is preferably a silica gel column to obtain a pure product of the 3, 4-oxygen-ring selenic acid galactose allyl glycoside. In the step of synthesizing the 3, 4-O-cyclo-selenious acid galactose allyl glycoside, the yield can reach more than 90 percent.
The invention uses acryloyl chloride to react with polysaccharide to prepare acrylic polysaccharide. The polysaccharide is water-soluble natural polysaccharide containing 6-primary hydroxyl, such as beta-1-3-glucan, lentinan, schizophyllan, carrageenan polysaccharide with small molecular weight, chitosan oligosaccharide and the like. The polysaccharide is first dissolved in a solvent. The solvent is not particularly limited in the present invention, and DMF can be used for dissolving the polysaccharide. Adding NaHCO into polysaccharide solution3Solid sodium bicarbonate is preferred. The polysaccharide isThe molar ratio to sodium bicarbonate is preferably 1: 5 to 50, and more preferably 1:10 to 30. And (3) dropwise adding acryloyl chloride into the polysaccharide solution, wherein the molar ratio of the polysaccharide to the dropwise added acryloyl chloride is preferably 1:1 to 50, more preferably 1:10 to 40, and still more preferably 1:20 to 30. And maintaining the temperature of the reaction liquid not to exceed 40 ℃ in the dropping process, and more preferably 15-35 ℃. Preferably, the reaction is completed within 2 to 4 hours after the dropwise addition is stopped. And pouring the reaction liquid into cold water, and adding cold ethanol to precipitate the acrylated polysaccharide, wherein the addition amount of the cold ethanol is preferably 3-7 times of the volume of the reaction liquid. The recovery rate of the synthesized acrylated polysaccharide is more than 95 percent.
The acrylated polysaccharide and 3, 4-oxygen-cyclo-selenic acid galactose allyl glycoside are subjected to a displacement reaction under the action of a Ru catalyst to obtain the esterified selenium polysaccharide. The Ru catalyst is not particularly limited in the present invention, and in the specific examples of the present invention, a Grubbs-I generation Ru catalyst is preferable. The above-mentioned substitution reaction of the present invention is preferably carried out in a DMF solvent, preferably by dissolving the acrylated polysaccharide in DMF, adding 3, 4-O-galactoallylselenite glycoside, and finally adding a catalytic amount of Ru catalyst. In the present invention, the molar ratio of the acrylated polysaccharide to the 3, 4-O-cyclo-selenic acid galactoallylglycoside is preferably 1:1 to 4, more preferably 1:2 to 3, and the amount of the Ru catalyst added is preferably 0.1 to 0.5 equivalent. The invention can complete the olefin replacement reaction within 4-5 hours, namely, the organic selenium sugar unit is combined on the polysaccharide. Dispersing the reaction solution in a water phase, and precipitating the product by using ethanol with 4-5 times of volume at low temperature. The product can be purified by repeating water-soluble ethanol precipitation, and the yield is 80-95%.
The invention also comprises the esterified selenium polysaccharide prepared by the preparation method, and the structural formula of the esterified selenium polysaccharide is as follows:
wherein m is a selenylated saccharide unit, and n is the number of saccharide units of the natural polysaccharide. The content of the organic selenium in the esterified selenium polysaccharide prepared by the invention can reach 1 to 10 ppm.
The esterified selenium polysaccharide has the potential of improving the blood selenium content and various immune functions, and can be used for improving the immune function in foods, medicines or health-care products.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
The following examples are provided to illustrate the present invention, and the polysaccharide is commercially available water-soluble β -1-3-glucan, but the scope of the present invention is not limited thereto.
Example 1
At 0 ℃, under the protection of nitrogen, 1 equivalent of O ═ SeCl2Dropwise adding the mixture into a commercial D-galactose allyl glycoside DMSO solution containing 4 equivalents of triethylamine, reacting at room temperature for 4 hours, pouring an aqueous phase, extracting with ethyl acetate, washing with saturated saline solution, drying an organic phase with anhydrous sodium sulfate, evaporating to dryness on a rotary evaporator under reduced pressure, and performing silica gel column chromatography to obtain a pure product with the yield of more than 90%.
Suspending 100mg of beta-1-3-glucan in 10 ml of anhydrous DMF, adding 5 equivalents of solid sodium bicarbonate and 1 equivalent of acryloyl chloride, keeping the temperature of the reaction solution not to exceed 40 ℃ for 2 hours, pouring the reaction solution into cold water while stirring, adding 4 times of cold ethanol in volume to precipitate an intermediate product, and freeze-drying the intermediate product for later use. The intermediate is resuspended in dry DMF, 1 equivalent of 3, 4-O-Cycloselenic acid galactoallylglycoside is added, then 0.1 equivalent of Grubbs-I Ru catalyst is added, the organoselenium polysaccharide is poured into the aqueous phase for 4 hours, and the product is precipitated with 4 volumes of ethanol at low temperature. The selenium content was 1.8 ppm by ICPMS analysis.
Example 2
At 0 ℃, under the protection of nitrogen, 5 equivalents of O ═ SeCl2Dropwise adding into D-galactose allyl glycoside DMF solution containing 2 equivalents of triethylamine, reacting at room temperature for 2 hr, pouring into water phase, extracting with ethyl acetate, washing with saturated saline solution, and collecting organic phase with anhydrous sulfurDrying sodium salt, evaporating to dryness under reduced pressure on a rotary evaporator, and performing silica gel column chromatography to obtain pure product with yield higher than 90%.
Suspending 100mg of beta-1-3-glucan in 20 ml of anhydrous DMF, adding 50 equivalents of solid sodium bicarbonate and 50 equivalents of acryloyl chloride, keeping the temperature of the reaction solution not to exceed 40 ℃ for 4 hours, pouring the reaction solution into cold water while stirring, adding 6 times of cold ethanol in volume to precipitate an intermediate product, and freeze-drying the intermediate product for later use. The intermediate is resuspended in dry DMF, 50 equivalents of 3, 4-O-cyclo-selenic acid galactosyl allyl glycoside are added, then 0.5 equivalent of Grubbs-I Ru catalyst is added, after 5 hours of reaction, the organic selenium polysaccharide is poured into water phase, and the product is precipitated by 5 times volume of ethanol at low temperature. The selenium content was 9.6 ppm by ICPMS analysis.
Example 3
At 0 ℃, under the protection of nitrogen, 3 equivalents of O ═ SeCl2Dropwise adding the mixture into a D-galactose allyl glycoside DMF solution containing 3 equivalents of triethylamine, reacting at room temperature for 3 hours, pouring an aqueous phase, extracting with ethyl acetate, washing with saturated saline solution, drying an organic phase with anhydrous sodium sulfate, evaporating to dryness on a rotary evaporator under reduced pressure, and performing silica gel column chromatography to obtain a pure product, wherein the yield is more than 90%.
Suspending 100mg of beta-1-3-glucan in 20 ml of anhydrous DMF, adding 20 equivalents of solid sodium bicarbonate and 20 equivalents of acryloyl chloride, keeping the temperature of the reaction solution not to exceed 40 ℃ for 4 hours, pouring the reaction solution into cold water while stirring, adding 5 times of cold ethanol in volume to precipitate an intermediate product, and freeze-drying the intermediate product for later use. The intermediate is resuspended in dry DMF, 20 equivalents of 3, 4-O-cyclo-selenic acid galactosyl allyl glycoside are added, then 0.2 equivalent of Grubbs-I Ru catalyst is added, after 4 hours of reaction, the organic selenium polysaccharide is poured into the water phase, and the product is precipitated by 4 times volume of ethanol at low temperature. The selenium content was 4.6 ppm by ICPMS analysis.
Example 4
B-6 splenocytes are taken as an experimental model, carrageenan, chitohexaose, 1, 3-glucohexaose and PMA + innomycin are taken as a reference substance, and the reference substance is incubated in a culture solution containing esterified selenium polysaccharide (2-5 micrograms of selenium/mL) for 48 hours to detect the expression quantity of IL-4 and IFN-gamma.
The culture solution for culturing B-6 splenocytes is a culture solution for culturing B-6 splenocytes in the field, and comprises essential and non-essential amino acids, vitamins, glucose, hormones, growth factors, trace minerals, low-concentration fetal calf serum (2%) and 5ml of penicillin/streptomycin solution. The buffer system of the culture medium is phosphate buffer saline solution PBS containing 5% CO2The pH value after equilibration in the cell culture box of (1) was 7.4.
After the cells were harvested, they were stained with the Cytofix/Cytoperm kit from BD Bioscience and detected by flow cytometry, and the detection results are shown in FIG. 1. Compared with 1, 3-glucose hexaose, the maximum expression level of IL-4 of B-6 splenocytes treated by the esterified selenium polysaccharide can be obviously improved by 60 percent, and the maximum expression level of IFN-gamma is improved by 36 percent, which shows that the esterified selenium polysaccharide can effectively improve the immunological competence of a system.
Example 5
By taking a mouse as a model, taking polysaccharide without esterified selenium as a control, continuously feeding the esterified selenium polysaccharide prepared by the invention at a selenium dose of 2-5 micrograms of selenium/day for 2 weeks, continuously carrying out blood detection from the third week to the sixth week (at the moment, the esterified selenium is still fed, the mode dose is the same as the former dose), finding once per week that the selenium content in serum is maximally improved by more than 30%, the red blood cells are improved by 20%, the white blood cells are increased by 10%, and beginning at the fifth week, the detection data tend to be stable.
TABLE 1 blood test results at different time periods after feeding mice
Example 6
The chicken was used as a model, the esterified selenium polysaccharide of the present invention was continuously fed at a dose of 5 micrograms selenium/day after the birth of the chicken using the polysaccharide without esterified selenium as a control, and the activity detection of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) was performed at 7 days, 14 days, 21 days, and 28 days after the feeding (using a commercially available kit EnzyChrom)TMGlutahione peroxiaseassay Kit and MlBio (mouse superoxide dismutase (SOD) reagent)The cartridge completes the test). The results show that the GHS-Px activity of the chicks reaches the maximum value in 14-21 days, the GHS-Px activity is improved by 15%, and the SOD reaches the maximum value in 7-14 days, the GHS-Px activity is improved by 12%, which indicates that the esterified selenium polysaccharide has better antioxidant function.
TABLE 2 Activity of GSH-Px and SOD in different periods of time for feeding chicks
(U/mg)
|
Day 1
|
Day 7
|
Day 14
|
Day 21
|
Day 28
|
Serum GSH-Px
|
1869
|
1879
|
1975
|
2150
|
2090
|
Serum SOD
|
180
|
191
|
195
|
190
|
187 |
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.